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MULTIMODAL EVALUATION OF ESOPHAGEAL
EPITHELIAL RESPONSE IN
GASTROESOPHAGEAL REFLUX DISEASE
Ph.D. Thesis
Dorottya Laczkó M.D.
Supervisors:
Viktória Venglovecz Ph.D., Department of Pharmacology and Pharmacotherapy,
University of Szeged
András Rosztóczy M.D. Ph.D., First Department of Medicine, University of Szeged
Department of Pharmacology and Pharmacotherapy,
First Department of Medicine,
University of Szeged, Szeged
Hungary
2016
- 2 -
TABLE OF CONTENTS
1. LIST OF ABBREVIATIONS - 4 -
2. LIST OF FULL PAPERS CITED IN THE THESIS - 5 -
3. LIST OF FULL PAPERS NOT RELATED TO THE THESIS - 5 -
4. SUMMARY - 6 -
5. INTRODUCTION - 8 -
5.1. Definition and epidemiology of GERD - 8 -
5.2. Classification and clinical manifestation of GERD - 8 -
5.2.1. NERD - 9 -
5.2.2. ERD - 9 -
5.2.3. BE - 9 -
5.3. Pathogenesis of GERD - 9 -
5.3.1. The role of ion transporters in the pathogenesis of GERD - 10 -
5.3.2. The role of autophagy in the pathogenesis of GERD and BE - 11 -
5.3.3. Modeling the pathogenesis of GERD - 12 -
6. AIMS - 13 -
7. MATERIALS AND METHODS - 14 -
7.1. Cell lines - 14 -
7.2. 3-dimensional organotypic culture (3D OTC) - 14 -
7.3. Immune cells and cytokine treatment - 15 -
7.4. Patients - 16 -
7.5. Chemicals and solutions - 16 -
7.6. Measurement of pHi and [Ca2+]i with microfluorimetry - 17 -
7.7. Determination of buffering capacity and base efflux - 18 -
7.8. Measurement of the activity of NHE, NBC and CBE - 18 -
7.9. Bile acid treatments - 19 -
7.10. RNA isolation, reverse transcription and quantitative real-time PCR - 19 -
7.11. Western Blot analysis - 20 -
7.12. Immunohistochemistry - 20 -
7.13. TUNEL assay - 21 -
7.14. Flow cytometry analysis - 21 -
- 3 -
7.15. Statistical analysis - 21 -
8. RESULTS - 22 -
8.1. Role of ion transporters in the bile acid-induced esophageal injury - 22 -
8.1.1. The main pHi regulatory mechanisms in human EECs - 22 -
8.1.2. Bile acids induces Ca2+ release via activation of IP3-mediated pathway - 26 -
8.1.3. Acute effects of bile acids on the activity of ion transporters in EECs - 28 -
8.1.4. Chronic exposure of EECs to bile acids - 30 -
8.2. Role of autophagy in the pathogenesis of GERD and BE - 33 -
8.3. Modeling esophagitis using 3D organotypic culture system - 37 -
8.3.1. The organotypic culture environment sustains immune cells and permits their normal
activation when stimulated by cytokines - 37 -
8.3.2. Inflammatory OTC environment induces changes in epithelial morphology - 40 -
8.3.3. The pro-inflammatory environment modeled with the OTC culture system alters cell
proliferation and cell death in the epithelium - 41 -
9. DISCUSSION - 43 -
10. CONCLUSIONS AND NEW RESULTS - 49 -
11. ACKNOWLEDGEMENTS - 50 -
12. REFERENCES - 51 -
1. LIST OF ABBREVIATIONS
2D: 2-dimensional
3D: 3-dimensional
7AAD: 7-amino actinomycin D
AV: autophagic vesicle
BAC: bile acid cocktail
BE: Barrett’s esophagus
BCECF-AM: 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester
[Ca2+]i: intracellular Ca2+concentration
CBE: Cl-/HCO3 – exchanger
CQ: chloroquine
DCF: 2’,7’-dichlorodihydrofluorescein diacetate
EAC: esophageal adenocarcinoma
EECs: esophageal epithelial cells
GERD: gastroesophageal reflux disease
IFN-γ: interferon-γ
IHC: immunohistochemistry
IRF-1: interferon regulatory factor-1
IL: interleukin
pHi: intracellular pH
NHE: Na+/H+ exchanger
NBC: Na+/HCO3– cotransporter
OTC: organotypic culture
PBMC: peripheral blood mononuclear cell
ROS: reactive oxygen species
SE: squamous epithelium
- 5 -
2. LIST OF FULL PAPERS CITED IN THE THESIS
I. Dorottya Laczkó, András Rosztóczy, Klaudia Birkás, Máté Katona, Zoltán
Rakonczay Jr., László Tiszlavicz, Richárd Róka, Tibor Wittmann, Péter Hegyi,
Viktória Venglovecz. Role of ion transporters in the bile acid-induced
esophageal injury. American Journal of Physiology-Gastrointestinal and Liver
Physiology 2016 Jul 1;311(1):G16-31. IF (2015): 3.798
II. Jianping Kong*, Kelly A. Whelan*, Dorottya Laczkó, Brendan Dang, Angeliz
Caro Monroig, Ali Soroush, John Falcone, Ravi K. Amaravadi, Anil K. Rustgi,
Gregory G. Ginsberg, Gary W. Falk, Hiroshi Nakagawa, John P. Lynch.
Autophagy levels are elevated in Barrett's esophagus and promote cell survival
from acid and oxidative stress. Molecular Carcinogenesis 2016
Nov;55(11):1526-1541. IF (2015): 4.808 (*these authors contributed equally)
III. Dorottya Laczkó, Fang Wang, F. Bradley Johnson, Nirag Jhala, András
Rosztóczy, Gregory G. Ginsberg, Gary W. Falk, Anil K. Rustgi, John P. Lynch:
Modeling esophagitis using human 3D organotypic culture system. American
Journal of Pathology (Under revision)
3. LIST OF FULL PAPERS NOT RELATED TO THE THESIS
IV. Andrea Szentesi, Emese Tóth, Emese Bálint, Júlia Fanczal, Tamara Madácsy,
Dorottya Laczkó, Imre Ignáth, Anita Balázs, Petra Pallagi, József Maléth,
Zoltán Rakonczay Jr, Balázs Kui, Dóra Illés, Katalin Márta, Alexandra
Demcsák, Andrea Párniczky, Gabriella Pár, Szilárd Gódi, Dóra Mosztbacher,
Ákos Szücs, Adrienn Halász, Ferenc Izbéki, Nelli Farkas, Péter Hegyi: Analysis
of Research Activity in Gastroenterology: Pancreatitis is in Real Danger Plos
One 2016 (Accepted for publication PONE-D-16-26557R1) IF (2015): 3.057
Number of full publications: 3 (1 first author)
Cumulative impact factor: 11.663
- 6 -
4. SUMMARY
Background & Aims: In gastroesophageal reflux disease (GERD) gastric acid and bile reflux into
the esophagus causing symptoms and/or complications. GERD is a significant condition of the
gastrointestinal tract, affecting nearly 20% of the population of the Western countries. The
refluxate can cause tissue injury and trigger an inflammatory response. GERD and the resulting
subsequent inflammation are risk factors for the development of esophageal strictures, Barrett’s
esophagus (BE), and esophageal adenocarcinoma (EAC). Therefore, the investigation of the
pathogenesis of GERD is of critical importance. The aims of our studies:
I. To investigate the role of ion transport mechanisms in the esophageal mucosal defense
against bile acid-induced cellular injury, in Barrett’s derived esophageal epithelial cells
II. Characterize the functional role of autophagy in a wide range of esophageal epithelial
cell lines from normal squamous to Barrett’s and adenocarcinoma cells
III. To modify the newly developed, innovative multicellular 3-dimensional (3D)
organotypic culture system (OTC) to model inflammatory conditions observed in
patients with reflux disease.
Methods:
I. In order to study ion transport mechanisms, non-dysplastic (CP-A) and dysplastic (CP-
D) human Barrett’s derived cell lines were acutely exposed to bile acid cocktail (BAC)
and the changes in intracellular pH (pHi) and Ca2+ concentration ([Ca2+]i) were
measured by microfluorometry. mRNA and protein expression of ion transporters were
investigated by qPCR, Western blot, and immunohistochemistry (IHC) in cell lines and
human esophageal biopsy samples.
II. To demonstrate a functional role for autophagy, normal squamous (STR), Barrett’s
derived (CP-A and CP-D), and adenocarcinoma (OE19) cell lines were exposed to an
acid pulse (pH=3.5) followed by incubation in the presence or absence of chloroquine
(CQ), an autophagy inhibitor and the level of reactive oxygen species (ROS),
autophagy level and cell survival were measured by flow cytometry.
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III. OTC system is a novel engineered 3D human tissue reconstruction in which human
esophageal fibroblasts are embedded in a collagen matrix with human esophageal
epithelial cells (EECs) seeded on top. To model esophagitis peripheral blood
mononuclear cells (PBMCs) were included in the collagen matrix. After 15 days OTCs
were harvested for histological analysis, RNA and protein isolation. Cultures were then
screened for changes in epithelial morphology, cell proliferation and apoptosis.
Results and Conclusions:
I. We have identified the presence of a Na+/H+ exchanger (NHE), Na+/HCO3−
cotransporter (NBC), and a Cl−-dependent HCO3− secretory mechanism in CP-A and
CP-D cell lines. Acutely administered BAC stimulated HCO3− secretion in both cell
lines and the NHE activity in CP-D cells by an inositol triphosphate-dependent Ca2+
release. Chronic treatment of the cells increased the expression of ion transporters
compared to non-treated cells. A similar expression pattern was observed in biopsy
samples from BE compared with normal squamous epithelium (SE). We speculate that
these adaptive processes of the metaplastic cells represent an important mucosal
defense against the bile acid-induced epithelial injury.
II. Pharmacologic inhibition of autophagy by CQ following acid stress has increased the
level of ROS in STR and CP-A cells and diminished cell survival in all cell lines. Our
findings provide new evidences that autophagy may have an important contribution to
the pathogenesis and progression of BE.
III. Addition and activation of immune cells in the OTC system induced apoptosis as well
as a regenerative response in the epithelial cells, as has been seen in human GERD
pathology. These findings support the concept that OTC can be adapted to model
inflammatory conditions in GERD and to understand better the pathophysiology of the
disease.
In summary, in this Ph.D. thesis we tried to give a better insight into the cellular and molecular
events in GERD. We believe that our studies may provide new possibilities to develop new
strategies in the treatment of GERD and BE.
- 8 -
5. INTRODUCTION
5.1. Definition and epidemiology of GERD
GERD is one of the most common disorder of the gastrointestinal system. It refers to a
condition where the retrogate flow of gastric and duodenal content provokes symptoms and/or
complications. (1, 2) According to population-based studies, GERD-related symptoms are
extremely common in adults and they are sufficient to impair significantly the health-related
quality of life. (3) Weekly occuring reflux-related symptoms are reported by nearly 20% of the
population in developed Western countries. (4) Moreover their incidence and prevalence increases
in such parts of the world where they were previously uncommon, particularly in South-East Asia
and the Far East. (1, 5)
5.2. Classification and clinical manifestation of GERD
GERD is divided into three phenotypic subcategories: non-erosive GERD (NERD), erosive
GERD (ERD) and Barrett’s esophagus (BE) based on the endoscopic evaluation of the esophagus.
Although some authors considered GERD as a spectrum disease, which is caracterized by the
gradual progression from the mildest non-erosiv form to the most severe stages (stricture,
adenocarcinoma) data support better the more recent phenotypic subgroup theory of Fass, since a
little if any transformation can be detected between them. (6)
GERD can be classified based on the presence or absence of reflux related typical or
atipical symptoms according to the Montreal criteria as well. (7) In contrast to the instrumental
(endoscopy + esophageal function testing) diagnosis this latter has the advantage of considering
troublesome symptoms, although asymptomatic GERD patients (silent GERD) may not be
recognized by this system. This is particularly important in BE where a significant proportion of
the patients have not reflux related symptoms at all (8)
The typical GERD symtoms are heartburn and/or acid regurgitation. In addtition, GERD
can present with other less typical manifestations like cardiac chest pain, chronic cough, ear, nose
and throat symptoms. Interestingly, NERD patients do not neccesserely have excesscive amount
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of acid regurgitation (9) and the classical anatomical alterations like hiatal hernia is less prevelant.
(10)
5.2.1. NERD
Nearly 60% of GERD patients fall into this category. NERD refers to a condition where
pathologic reflux is present in the absence of endoscopically visible mucosal injury. However,
ultrastructural abnormalities are demonstrated at this stage of the disease as well evidenced by the
dilatation of intercellular spaces which can give a possible explantion for the development of the
symptoms. (11)
5.2.2. ERD
35% of GERD are presented as ERD. These patients have macroscopically visible mucosal
injury during upper endoscopy. Although most patients have non progressive mild to moderate
forms a minority may progress and end up in severe complications such as esophageal perforation,
bleeding or esophageal stirctures. (12)
5.2.3. BE
BE refers to the replacement of the multilayered SE with specialized intestinal type of
columnar epithelium at the distal part of the esophagus. (13) Although the exact pathomechanism
of BE is still unknown the major pathogenetic factors considered to be the excessive acid and bile
exposure and the subsequent chronic inflammation triggered by the chemical injury. (14) The
presence of Barrett-metaplastic tissue supposed to be an adaptive response to provide a better
protection against chemical injury. (15) However, BE is associated with a 30- to 40-fold increased
risk for the development of EAC, (16) a cancer whose incidence has rapidly increased in the past
few decades in Western countries. (17) Based on this observation BE is considered to be the most
severe complication of GERD, therefore its investigation and surveillence is necessary. (18, 19)
5.3. Pathogenesis of GERD
Nearly all healthy individuals experience some sort of physiological reflux, which is
generally short in duration and occurs infrequently, most typically in the postprandial period. (20)
The refluxate may contain numerous substances such as bile acids and pancreatic enzymes, not
- 10 -
only hydrochloric acid and pepsin. Under phyisological conditions, these injurious noxas are
abolished by the defensive mechanisms of the esophagus. However, in patients with GERD, there
is an imbalance between these aggressive and protective factors. Gastric acid is considered to be
the most important contributing factor to GERD related symptoms and complications. Its
pathogenic role is supported by the fact that GERD patients experience significantly increased
acidic reflux (21) and acid supressing therapy has brought relief to GERD patients. (22) However,
the severity of the disease cannot be explained by the gastric acid exposure alone. (21)
Duodenogastric reflux contains high concentration of bile acids which have been shown to have
carcinogenic properties by inducing epithelial proliferation and reducing the rate of apoptosis in
EECs. (23) Moreover, the level of biliary reflux was not only shown to be increased in patients
with specialized intestinal metaplasia (SIM) compared to those without SIM, but further significant
increase has been demostrated in those with histological signs of dysplasia. (21) Bile acids were
also shown to have a fundamental role in the development of columnar metaplasia by inducing the
expression of CDX2 transciptional factor which is responsible for normal intestinal differentiation.
(24, 25) Thus, bile acids are considered to be the other crucial pathogenic factor in the pathogensis
of GERD related esophageal injury, especially BE and EAC. (22). To date, most papers have
focused on the investigation of the direct effects of gastric acid and bile on EECs. (26-30).
However, an alternative hypothesis has recently been emerged suggesting that beside the direct
chemical challenge, cytokine-mediated inflammatory responses might have at least similarly
important contribution in the development of esophageal mucosal injury in patients with GERD.
(31, 32)
5.3.1. The role of ion transporters in the pathogenesis of GERD
Under physiological conditions highly efficient barriers exist in the esophagus which can
protect esophageal epithelium from harmful compounds. Epithelial resistance, which was first
described by Orlando et al. is one of the key element of the defense. (32) Esophageal resistance
can be divided into three functional categories: the pre-epithelial, epithelial and post-epithelial
defense. Pre-epithelial protection is consist of unstirred water layer and mucous layer with HCO3-
in it. The main role of this defense mechanism is to maintain a substantial pH gradient from lumen
to surface cells. Post-epithelial defense refers to the adaquate blood supply of the tissue which
plays an essential role in the regulation of interstitial pH through removal of acidic byproducts.(33)
- 11 -
Epithelial defense is the most important component of esophageal resistence. It has both
structural and functional components and the transport proteins on the apical and basolateral
membranes of EECs play a crucial role in it. (33, 34) At the apical membrane of EECs, only a
nonselective cation channel has been identified so far. (35) This channel is present in the SE of
rabbits and has been shown equally permeable to Na+, Li+, K+, or even H+. The physiological role
of this channel in esophageal epithelial function is poorly understood. Tobey et al. (36) have shown
that acidic pH inhibits channel activity so H+ cannot enter the cell through this channel and
therefore may represent a protective mechanism against luminal acidity. Others suggest that this
cation channel plays a role in cell differentiation. Blockade of this channel by acidic pH may inhibit
the replenishment of polarized epithelial cells from undifferentiated basal cells. (35)
In contrast, at the basolateral membrane of SE several ion transporters have been identified.
Tobey et al. (36) have shown the presence of a Na+-dependent and Na+-independent, disulfonic
stilbene-sensitive, Cl−/HCO3− exchangers (CBE) on cultured rabbit SE. (37, 38) The Na+-
independent CBE mediates the efflux of HCO3− into the lumen, which results in the acidification
of the pHi. In contrast, the Na+-dependent CBE operates in a reverse mode and promotes the influx
of HCO3− in exchange for intracellular Cl− and therefore contributes to the alkalization of the cell.
(37, 38) Beside the CBEs, an amiloride-sensitive Na+/H+ exchanger (NHE) has also been identified
on the basolateral membrane of rat, rabbit and human SE. (37, 39, 40) Among the nine known
NHE isoforms, NHE1 has been shown to be present on EECs using reverse-transcription-PCR and
Western blot. The major role of NHE1 in the esophagus is the regulation of pHi by the
electroneutral exchange of intracellular H+ to extracellular Na+. In addition, NHE1 is also
important in several defensive mechanisms such as cell volume regulation, proliferation, and cell
survival. (41-43)
These studies have been performed on normal esophageal epithelium; however, the activity
or expression of ion transporters in the columnar epithelia under pathophysiological conditions is
less characterized. (44)
5.3.2. The role of autophagy in the pathogenesis of GERD and BE
Autophagy is a lysosome dependent cellular mechanism which degrades the damaged or
obsolete organelles and proteins in the cells. (45) Basal level of autophagy has a fundamental
physiological role in cellular homeostasis like development, immune defense, programmed cell
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death and prevention of neuron degradation. (45) Furthermore, autophagy is induced as an adaptive
response to cellular stress, either from nutrient/growth factor deprivation, hypoxia, oxidative
stress, accumulation of protein aggregates, and endoplasmic reticulum stress. (46, 47) During
autophagy the potentially toxic cytoplasmic constituents are encapsulated by pre-autophagosomal-
structures called “phagophores” that mature into double-membrane vesicles called
“autophagosomes” and fuse with lysosomes where the content is degraded. (48)
In normal tissues, autophagy is responsible for maintaining cell homeostasis and prevents
carcinogenesis (49) by inducing apoptosis. (48, 50, 51) Therefore, the dysregulation of autophagy
has been linked to a growing list of diseases such as eosinophilic esophagitis (52), inflammatory
bowel disease (53) and Parkinson’s disease (54). Autophagy can also act as a double edged sword
in the cells: once cancer develops, many cancer cells upregulate autophagy to survive hypoxia and
nutrient limitation. (48) Interestingly, despite the growing evidence between cancer and
autophagy, the importance of autophagy in pre-cancerous lesions like BE is less established.
Furthermore, in GERD, the gastric acid and bile contribute to the inflammation and cellular stress
of esophageal epithelium which are important activators of autophagy. (27, 55-57) In contrast, to
date there is only a single publication on the role of autophagy in BE mainly focusing on the role
of Beclin1 autophagy gene only. (58)
5.3.3. Modeling the pathogenesis of GERD
Research into GERD and its complications has been restricted by the availability of suitable
laboratory approaches to model these conditions. Much of the past work has relied upon the
availability of human patient biopsies, which are difficult to obtain and primarily suitable for
descriptive studies. Immortalized cell lines, representing normal squamous, Barrett’s, and EAC
are available (59-62), however 2D cultured cells can not effectively model complex interactions
between epithelial cells and their microenvironment. (14) Animal models are another important
tool of biomedical research. However, animal models for the diseases of the esophagus are limited
as well, in part due to anatomic differences between mice and humans at the squamo-columnar
junction. (63) OTC system is an innovative multicellular system which attempts to better model
human tissues. (64) Under 3D OTC conditions, human esophageal keratinocytes undergo a
complete differentiation and stratification producing a fully mature epithelium. Its advantage
relates to the normal polarization and differentiation of cells and gene expression patterns are
- 13 -
similar to the in vivo conditions. OTC is an in vitro tool which is still physiologically relevant and
helps to understand better the complex interactions between epithelial cells and their
microenvironment.
6. AIMS
The specific aims of our studies:
I. In the first part of the thesis we attempted to identify the ion transport mechanisms in
columnar epithelial cells derived from Barrett's metaplasia and to characterize the
effect of main internal risk factors (such as HCl, bile acids) on these transporters.
Furthermore, we aimed to compare their mRNA and protein expression profile of ion
transporters with human squamous and columnar epithelial cells obtained from
endoscopic biopsies of normal esophageal mucosa and BE.
II. In the second part of the thesis, we planned to explore the functional role of the
autophagic response in cellular oxidative stress and cell survival in esophageal cells
representing different severity stages of adenocarcinoma in an in vitro model of acid
reflux.
III. Finally, our last objective was to establish whether OTC could be applied as a novel
platform for the study of inflammatory environment in GERD.
- 14 -
7. MATERIALS AND METHODS
7.1. Cell lines
Immortalized human squamous EECs (STR, University of Pennsylvania, Philadelphia, PA,
USA) were developed and maintained as previously described. (65) CP-A human, non-dysplastic
Barrett’s esophageal cell line was obtained from American Type Culture Collection (ATCC,
Manassas, VA, USA). CP-D human, dysplastic Barrett’s cell line was kindly provided by Peter
Rabinovich (University of Washington). CP-A and CP-D cells were adapted to serum-free
conditions in keratinocyte serum-free medium (KSFM, Invitrogen, Waltham, MA, USA). OE19
human adenocarcinoma cells were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and
maintained in RPMI 1640 with 2 mM glutamine and 10% fetal calf serum. Medium was replaced
in every 2 days on all cell lines. Cultures were continually incubated at 37 °C and gassed with the
mixture of 5% CO2 and 95% air. Lentiviral vector LC3-GFP was obtained from Dr Craig
Thompson (Memorial Sloan Kettering, New York, NY, USA) and transfected into 293T cells
along with the packaging plasmids using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA)
reagent following the manufacturer’s instructions. Then OE19 cells were exposed to virus in the
presence of 8 mg/mL polybrene for 16-18h respectively. LC3-GFP expressing cell populations
were isolated by sorted for GFP using flow cytometry. LC3-GFP expression was confirmed by
examination of the cells by confocal fluorescent microscopy on a Nikon Eclipse Ti-U microscope.
LC3-GFP+ vesicles were identified and quantified using the spot finder application in the Volocity
image analysis software package (Perkin Elmer, Waltham, MA, USA)
7.2. 3-dimensional organotypic culture (3D OTC)
The fibroblast feeder layer and 6.75x105 PBMCs were embedded within a collagen/Matrigel
matrix and was allowed to mature for 7 days, after which 5×105 epithelial cells were seeded on top
and allowed to grow confluence for another 4 days. On day 11, the culture media level is reduced
bringing the keratinocytes to air-liquid interface which stimulates epithelial differentiation into a
multilayer epithelium typical for the esophagus. On day 15, OTCs were harvested for histology,
RNA and/or protein isolation. (Fig.1.)
- 15 -
Figure 1. Establishment of organotypic culture (OTC) system (adapted from Kalabis et al. Nat. Protoc. 2012) (A)
Inserts are placed on 6-well plates. (B) In the first step, there is placement of an acellular collagen matrix on the bottom
of an insert, followed by the addition of a layer of esophageal fibroblasts embedded in collagen. They are cultured
initially for 7 days, allowing for fibroblast-mediated constriction of the collagen matrix. (C) On day 7, the EECs are
seeded on the top of the contracted matrix. (D) The epithelization medium (EPM) 1 of the OTC is changed every 2
days. (E) On day 11, the level of the medium (EPM2) is reduced, therefore the epithelium is exposed to air to create
a liquid-air interface (E), thereby promoting epithelial stratification and differentiation. (F) Finally, on day 15, the
resulting OTC is harvested for histology and RNA/protein analyses.
7.3. Immune cells and cytokine treatment
PBMCs were collected from healthy volunteers and isolated freshly by the Human
Immunology Core at the University of Pennsylvania. To model TH1 inflammatory environment
pro-inflammatory cytokines IL-7 (10 ng/mL, Cell Signaling, Beverly, MA, USA) and IL-15 (20
ng/mL, ProSpec Rehovot, Israel) were included to the cell culture media; IL-2 (10 U/mL, BD
Biosciences, San Jose, CA, USA) was also added to support PBMC viability. The cytokines were
replenished during each media change.
- 16 -
7.4. Patients
Fourteen patients with endoscopic evidence of esophageal metaplasia were enrolled in the
First Department of Medicine, University of Szeged, Hungary. Endoscopic procedures were
carried out by standard, high resolution, white-light endoscopes (Olympus GIF-Q165) and the
Prague C&M criteria were applied for the description of esophageal metaplasia. (66) Four biopsy
samples were obtained from the macroscopically visible metaplastic columnar epithelium of the
esophagus and another four from the normal squamous lining. Two of each sample were formalin-
fixed and submitted for histological evaluation including IHC. The remaining two samples were
immediately placed and stored in RNA-later solution for real-time PCR analysis at -20°C. All
procedures were performed with informed patient consent and under approved human subject’s
protocols from University of Szeged (No.: 2348).
7.5. Chemicals and solutions
General laboratory chemicals and bile acid salts were obtained from Sigma-Aldrich
(Budapest, Hungary). 2,7-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester
(BCECF-AM), 2-(6-(bis(carboxymethyl)amino)-5-(2-(2-(bis(carboxymethyl)amino)-5-
methylphenoxy)ethoxy)-2-benzofuranyl)-5-oxazolecarboxylic acetoxymethyl ester (Fura-2 AM),
1,2-bis(o-aminophenoxy)ethane-N,N,N9,N9-tetraacetic acid (BAPTA-AM), 4,4'-
diisothiocyanatodihydrostilbene-2,2'-disulfonic acid, disodium salt (H2DIDS) were from
Molecular Probes Inc (Eugene, OR, USA). BCECF-AM (2 µmol/l) and BAPTA-AM (40 µmol/l)
were prepared in dimethyl sulfoxide (DMSO), whereas FURA-2-AM (5 µmol/l) was dissolved in
DMSO, containing 20% pluronic acid. 4-isopropyl-3-methylsulphonylbenzoyl-guanidin
methanesulphonate (HOE-642) was provided by Sanofi Aventis (Frankfurt, Germany) and was
dissolved in DMSO. Nigericin (10 mM) was prepared in ethanol and stored at -20 ºC.
The compositions of the solutions used are shown in Table 1. Standard HEPES-buffered solutions
were gassed with 100% O2 and their pH was set to 7.4 with NaOH. Standard HCO3-/CO2-buffered
solutions were gassed with 95% O2/5% CO2 to set pH to 7.4. All experiments were performed at
37 ºC.
- 17 -
Table 1.: Composition of solutions. Values are in mM.
7.6. Measurement of pHi and [Ca2+]i with microfluorimetry
150.000-250.000 cells were seeded to 24 mm cover slips which were mounted on the stage
of an inverted fluorescence microscope linked to an Xcellence imaging system (Olympus,
Budapest, Hungary). Cells were bathed with different solutions at 37oC at the perfusion rate of
5-6 ml/min. 6-7 cells/region of interests (ROIs) were examined in each experiments and one
measurement per second was obtained. In order to estimate pHi cells were loaded with the pH-
sensitive fluorescent dye, BCECF-AM for 20-30 min at room temperature. Cells were excited
with 490 and 440 nm wavelengths, and the 490/440 fluorescence emission ratio was measured
at 535 nm. The calibration of the fluorescent emission ratio to pHi was performed with the high-
K+-nigericin technique, as previously described. (67, 68) To determine the changes of ([Ca2+]i)
StandardHepes
StandardHCO3
-
NH4ClHepes
NH4ClHCO3
-
Na+-freeHepes
Cl--freeHepes
Cl--freeHCO3
-
NaCl 130 115 110 95
KCl 5 5 5 5 5
MgCl2 1 1 1 1 1
CaCl2 1 1 1 1 1
Na-Hepes 10 10
Glucose 10 10 10 10 10 10 10
NaHCO3 25 25 25
NH4Cl 20 20
Hepes 10
NMDG-Cl 10
Na-gluconate
140 140 115
Mg-gluconate
1 1
Ca-gluconate
6 6
K-sulfate 5 2.5
- 18 -
cells were incubated with FURA2-AM and pluronic acid for 50-60 min. For excitation, 340 and
380 nm filters were used, and the changes in [Ca2+]i were calculated from the 340/380
fluorescence ratio measured at 510 nm.
7.7. Determination of buffering capacity and base efflux
The total buffering capacity (βtotal) of cells was estimated according to the NH4+ prepulse
technique, as previously described.(69, 70) Briefly, EECs were exposed to various concentrations
of NH4Cl in a Na+- and HCO3--free solutions. The total buffering capacity of the cells was
calculated using the following equation: βtotal = βi + βHCO3- = βi + 2.3 x [HCO3-]i, where βi refers to
the ability of intrinsic cellular components to buffer changes of pHi and was estimated by the
Henderson–Hasselbach equation. βHCO3- is the buffering capacity of the HCO3-/CO2 system. The
measured rates of pHi change (∆pH/∆t) were converted to transmembrane base flux J(B-) using
the equation: J(B-)=∆pH/∆t x βtotal. The βtotal value at the start point pHi was used for the calculation
of J(B-). We denote base influx as J(B) and base efflux (secretion) as -J(B-).
7.8. Measurement of the activity of NHE, NBC and CBE
In order to estimate the activity of NHEs, the NBC and CBE the NH4Cl prepulse technique
was used. Briefly, exposure of esophageal cells to 20 mM NH4Cl for 3 min induced an immediate
rise in pHi due to the rapid entry of lipophilic, basic NH3 into the cells. After the removal of NH4Cl,
pHi rapidly decreased. This acidification is caused by the dissociation of intracellular NH4+ to H+
and NH3, followed by the diffusion of NH3 out of the cell. In standard Hepes-buffered solution the
initial rate of pHi (ΔpH/Δt) recovery from the acid load (over the first 60 sec) reflects the activities
of NHEs, whereas in HCO3-/CO2-buffered solutions represents the activities of both NHEs and
NBC.(69)
Two independent methods have been performed in order to estimate CBE activity. Using
the NH4Cl prepulse technique the initial rate of pHi recovery from alkalosis in HCO3-/CO2
-buffered
solutions was analyzed.(69) Previous data have indicated that under these conditions the recovery
over the first 30 seconds reflects the activity of CBE. (69) The Cl- withdrawal technique was also
applied, where removal of Cl- from the external solution causes an immediate and reversible
alkalisation of the pHi due to the reverse operation of CBE under these conditions. Previous data
- 19 -
have shown that the initial rate of alkalisation over the first 60 seconds reflects the activity of CBE.
(71)
7.9. Bile acid treatments
In order to mimic the chronic bile acid exposure in GERD in vitro, cells were treated with
bile acid cocktail (BAC) at pH 7.5 and 5.5. Two days prior to bile acids treatment, cells were
seeded at 106 cells/75 cm2 tissue culture flasks and were grown to 70-80% of confluence. On the
second day, after the seeding, cells were treated with bile acids for 10 min pulses, 3 times a day up
to 7 days. The compostion of BAC was: 170 µM glycocholic acid (GC), 125 µM
glycochenodeoxycholic acid (GCDC), 100 µM deoxycholic acid (DC), 50 µM glycodeoxycholic
acid (GDC), 25 µM taurocholic acid (TC), 25 µM taurochenodeoxycholic acid (TCDC) and 8 µM
taurodeoxycholic acid (TDC). The composition and concentration of BAC mimics the bile acid
profile of GERD. (72-74)
7.10. RNA isolation, reverse transcription and quantitative real-time PCR
Total RNA was purified from individual cell cultures, biopsy samples and OTC epithelium
(after manually peeling off from the collagen base) using the RNA isolation kit of Macherey-Nagel
(Nucleospin RNA II kit, Macherey-Nagel, Düren, Germany) according to the manufacturer’s
instructions. The quantity of isolated RNA samples was checked by spectrophotometry (NanoDrop
3.1.0, Rockland, DE, USA). High-Capacity cDNA Archive Kit (Applied Biosystems Foster City,
CA, USA) was used for reverse transcription according to the manufacturer’s instructions. For
NHE-1, 2, NBC, Slc26a6 genes, reactions were performed on RotorGene 3000 instrument (Corbett
Research, Sydney, Australia) using Taqman probe sets (Applied Biosystems Foster City, CA,
USA). Hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene was used as an internal
control. For IRF-1 gene reaction was performed using the StepOne Plus instrument and the
amplifications were done using the SYBR Green PCR Master Mix (Applied Biosystems, Foster
City, CA, USA). GAPDH was used as the normalization control. In the case of cell lines, and OTC
epithelium the relative changes in gene expression were determined using the ΔΔCT method as
described in Applied Biosystems User Bulletin No. 2 (P/N 4303859). In the case of biopsy samples,
the relative expression values of NHE1, NHE2, NBC and SLC26a6 in normal and BE samples
- 20 -
was used to create box plots. In order to compare the expression of genes between normal and BE
samples, Wilcoxon test was used.
7.11. Western Blot analysis
20 μg of denatured protein was fractionated on a NuPAGE Bis-Tris 4–12% gel (Life
Technologies, Carlsbad, CA, USA). Following electrotransfer, Immobilon-P membranes
(Millipore, Billerica, MA, USA) were blocked with PBST containing 5% milk, followed by
overnight incubation with the following primary antibodies: rabbit anti-NHE1 (1:200, Alomone
Laboratories, Jerusalem, Israel) and IRF-1 (1:1000 Abcam, Cambridge, MA, USA) at 4oC. Mouse
anti-GAPDH (1:10000 Merck Millipore, Billerica, MA, USA) and mouse alpha tubulin (1:10000
Sigma-Aldrich, Saint Louis, MO, USA) were used as an internal control. Targeted proteins were
visualized using a chemiluminescence detection system (Amersham ECL or ECL Prime; GE
Healthcare Life Sciences, Pittsburgh, PA, USA)
7.12. Immunohistochemistry
5 μm paraffin-embedded sections of human, esophageal biopsy samples and OTCs were
deparaffinised, and heat mediated antigen retrieval was performed with 10 mmol/L citric acid
buffer (pH 6.0). Endogenous peroxidases were quenched using hydrogen peroxide before sections
were incubated in avidin D and biotin blocking reagents. Sections were incubated with primary
mouse monoclonal anti-NHE1 (1:100, Abcam, Cambridge, MA, USA), chicken anti-NHE2 (1:50,
Chemicon, Temecula, CA, USA), rabbit anti-CD45 (1:50, Abcam, Cambridge, MA, USA), rabbit
anti-Ki-67 (1:200, Abcam, Cambridge, MA, USA) antibodies and biotinylated secondary
antibodies and an avidin-horseradish peroxidase conjugate (Vectastain Elite ABC kit; Vector
Laboratories, Burlingame, CA, USA) following the manufacturer's protocol. The signal was
developed using the 3, 39-diaminobenzidine substrate kit (Vector Laboratories, Burlingame, CA,
USA). Sections were counterstained with hematoxylin. The specificity of the primary antibodies
was assessed by using mouse IgG1 or chicken IgY isotype controls. Slides were visualized under
Nikon E600 brightfield microscope (Melville, NY, USA) and digital images were taken with
iVision software (Atlanta, GA, USA). All images were analyzed with ImageJ software.
- 21 -
7.13. TUNEL assay
Apoptosis in paraffin-embedded sections was assessed using In Situ Cell Death Detection
Kit (Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions.
7.14. Flow cytometry analysis
To detect autophagy flux Cyto-ID (Enzo Life Sciences, Farmingdale, NY, USA) green
autophagy dye was used according to the manufacturer’s instruction. Cells were trypsinized into
single cells and stained with Cyto-ID for 30 min at 37oC prior to analysis. Intracellular levels of
ROS in epithelial cells were determined by flow cytometry with 2’, 7’-dichlorodihydrofluorescein
diacetate (DCF) dye (Life Technologies, Carlsbad, CA, USA). DCF is lipophilic and non-
fluorescent compound that is oxidized to fluorescent DCF by ROS, and is widely used to evaluate
cellular oxidative stress. For cell lines, cells were incubated with 10 µM DCF at 37°C for 30 min
and further cultured for up to 3h prior to analysis. OTCs were incubated with 10 µM DCF at 37°C
for 60 min and further cultured for up to 3 hours prior to peeling the epithelial layer and
trypsinizing into single cells. Cell death measures, at 24 h were carried out using staining with 7-
amino-actinomycin D (7AAD, Biolegend, San Diego, CA, USA). 7AAD fluoresces upon binding
to DNA and is normally excluded by living, intact cells. After cells were isolated from the culture
plate, washed, and centrifuged, they were resuspended in 0.5 mL of FACS buffer and 5ul of 7AAD
and further incubated for 5–10 min in the dark before the analysis and quantitation by flow
cytometry. Following stainings, cells were washed then analyzed in DPBS containing 1% BSA
using a FACS Calibur (BD Biosciences, San Jose, CA, USA). FlowJo software (Tree Star,
Ashland, OR, USA) was used for data analysis.
7.15. Statistical analysis
GraphPad Prism (San Diego, CA, USA) software was used for statistical analyses. Unpaired
student's t-test was used for comparisons between two groups. Data from multiple groups were
analyzed using one-way ANOVA with Tukey’s post-hoc test. All data were represented as mean
± standard error of the mean (SEM). p values ≤ 0.05 were accepted as significant.
- 22 -
8. RESULTS
8.1. Role of ion transporters in the bile acid-induced esophageal injury
8.1.1. The main pHi regulatory mechanisms in human EECs
In the first series of experiments, the resting pHi of CP-A and CP-D cell lines was calibrated
by the high K+/nigericin method. (68) Briefly, cells were exposed to standard HEPES solution (pH
7.4), followed by a 5-minutes incubations to a high K+/nigericin-Hepes solution at pH 7.28, 7.4
and 7.6. The classical linear model was used to determine the resting pHi of the cells. (67, 68) The
resting pHi levels of CP-A and CP-D were 7.32±0.03 and 7.31±0.03, respectively. The resting pHi
did not differ significantly among the pH experiments. (data not shown)
In the next step, the major functionally active ion transporters in Barrett’s derived cells
(CP-A and CP-D) were characterized. Na+-withdrawal from the standard Hepes-buffered solution
resulted in a rapid intracellular acidification (Fig. 2A) in CP-A cells which is likely due to the
inhibition of NHE. To further confirm the presence of NHE, NH4Cl prepulse technique was used
as it has been described in Materials and Methods. When Na+ was removed from the external
solution the recovery of pHi was completely abolished. (Fig. 2B) Similar results were confirmed
in CP-D cells, suggesting that these cells also possess functionally active NHE.
Figure 2. Investigation of NHE activity on CP-A cells. (A) Removal of Na+ from the standard HEPES solution
caused a rapid and marked intracellular acidosis in CP-A cells, which confirms the presence of a Na+-dependent H+
efflux mechanism. (B) Recovery from acid load reflects the activity of NHE in standard HEPES-buffered solution. In
the case of the second NH4Cl pulse, Na+ was removed from the external solution, 10 min before the pulse started,
during the NH4Cl pulse, and 10 min after the pulse.
A. B.
6,6
6,8
7,0
7,2
7,4
7,6
7,8
8,0
Hepes
Na+-free
3 min
pH
i
6,6
6,8
7,0
7,2
7,4
7,6
7,8
8,0
8,2
8,4
8,6
NH4Cl NH4Cl
3 min
Na+-free
Hepes
pH
i
- 23 -
To date, 9 members of the NHE family have been identified, all of which display different
regulation and expression pattern in the human body. Functional measurements were performed
to determine which NHE isoforms are present in CP-A and CP-D cells using the isoform selective
NHE inhibitor, HOE-642. At 1 µM, HOE-642 inhibits only NHE1, whereas at 50 µM inhibits both
NHE1 and NHE2. Using the NH4Cl prepulse technique it was shown that 1 µM HOE-642 inhibited
the recovery from acid load by 77.3 ± 3.0 % in CP-A and 70.0 ± 0.3 % in CP-D cells, whereas in
the presence of 50 µM HOE-642, the recovery was completely abolished in both cell lines. (Fig.
3A and B)
A
B
6,6
6,8
7,0
7,2
7,4
7,6
7,8
pH
i
Hepes
NH4Cl Na+-free
1 µM HOE-642 50 µM HOE-642
3 min
NH4Cl Na+-free NH4Cl Na+-free
* * **
N.D. N.D.
Figure 3. Investigation of different
NHE isoform activity on EECs. (A)
Representative pHi curve shows the
recovery from acid load in the
presence of 1 and 50 μM HOE-642.
(B) Summary data of the calculated
activities of the different NHE
isoforms in the presence of isoform
selective NHE inhibitor, HOE-642.
The rate of acid recovery [J(B−)] was
calculated from the ΔpH/Δt obtained
by linear regression analysis of pHi
measurements made over the first 60
s of recovery from the lowest pHi
level (start point pHi). The buffering
capacity at the start point pHi was
used for the calculation of J(B−).
N.D., not detected. Data are
presented as means ± SE. *P ≤ 0.05
vs. control; n = 15–25.
- 24 -
NBC represents another important pHi regulatory mechanism in several types of epithelial
cells (75-77). In the case of EECs, in standard HCO3-/CO2-buffered extracellular solution, the pHi
of CP-A cells rapidly decreased by the quick diffusion of CO2 into the cytoplasm. (Fig. 4A) A low
level of pHi recovery was found after acidosis, which is probably due to the influx of HCO3- into
the cells through NBC. Removal of Na+ resulted in the same level of acidification as in the standard
Hepes-buffered solution. (Fig. 4A) To further confirm the presence of NBC, the effect of H2DIDS,
an inhibitor of both NBC and CBE on the recovery from CO2-induced acidosis was investigated.
500 µM H2DIDS completely inhibited the regeneration from acidosis (Fig. 4B). However, after
the removal of H2DIDS from the external solution, the pHi completely recovered. Since CBE did
not affect the recovery from acidosis (see Fig. 4A), these results strongly suggest the presence of
functionally active NBC in CP-A cells. Using the same experimental protocol, the presence of
NBC was also confirmed in CP-D cells.
A B
Figure 4. Investigation of NBC activity on EECs. (A) Representative pHi curve showing the effect of Na+ removal
on CP-A cells in HCO3−/CO2-buffered solution. (B) Administration of 500 μM H2DIDS completely abolished the
recovery from acidosis in CP-A cells.
In order to estimate the activities of NHE and NBC, the effects of H2DIDS (500 µM) and
HOE-642 (50 μM) on the recovery from acid load was tested separately and together. Both
H2DIDS and HOE-642 equally reduced the recovery from acidosis, whereas combined
administration of these two agents completely abolished it. (Fig. 5A and B)
6,0
6,4
6,8
7,2
7,6
8,0
pH
i
Na+-free
HCO3-/CO2
3 min 6,5
6,9
7,3
7,7
8,1
8,5
pH
i
H2DIDS
HCO3-/CO2
3 min
- 25 -
A B
Figure 5. Investigation of NBC and NHE activities on EECs. (A) Representative pHi traces showing the effect of
H2DIDS (500 μM) and HOE-642 (50 μM) on the recovery from acidosis in HCO3−/CO2-buffered solution. CP-A cells
were acid loaded twice. The first NH4Cl pulse was the control and the second was the test. H2DIDS/HOE-642 was
added 1 min before the end of NH4Cl pulse and further 2 min after the pulse. (B) Summary data of the calculated NHE
and NBC activities. The rate of acid recovery [J(B−)] was calculated as described in Fig. 1. N.D. not detected. Data
are presented as means ± SE. *P ≤ 0.05 vs. control; n = 15–25.
Next we attempted to identify functionally active CBE. The activity of CBE was
investigated by the Cl- removal technique in the presence and absence of HCO3-/CO2. In the
absence of HCO3-, Cl- removal caused only a very low level and reversible alkalization. (Fig. 6A)
A B
Figure 6. Investigation of CBE activity on EECs. The activity of CBE was investigated by the Cl− removal technique
in the presence and absence of HCO3−/CO2. In standard HEPES-buffered solution (A), removal of Cl− (5 min) had no
significant effect on pHi. However, in standard HCO3−/CO2 solution (B), pHi in the absence of Cl− significantly
increased, indicating the presence of a functionally active CBE on CP-A cells.
6,0
6,2
6,4
6,6
6,8
7,0
7,2
7,4
7,6
7,8
8,0
8,2
pH
i
HCO3-/CO2
NH4Cl NH4Cl
3 min
H2DIDS/HOE-642
7,0
7,2
7,4
7,6
7,8
pH
i
Hepes
Cl- -free
1 min
6,5
6,8
7,1
7,4
7,7
8,0
pH
i
Cl- -free
HCO3-/CO2
1 min
- 26 -
However, removal of Cl− from the standard HCO3− /CO2 solution caused a marked and
reversible alkalization suggesting the presence of a Cl− dependent HCO3− efflux mechanism in CP-
A cells. (Fig. 6B) In case of CP-D cells, a marked alkalization was also observed after the removal
of external Cl- in the presence of HCO3-/CO2, suggesting that these cells also possess CBE.
8.1.2. Bile acids induces Ca2+ release via activation of IP3-mediated pathway
Bile acids are known activators of many intracellular processes including Ca2+ signaling
which is an important intracellular pathway. Therefore, in the next step we sought to identify the
effect of BAC on intracellular [Ca2+]i. Since bile acids have ionophore properties,(78, 79) their
effect was investigated both under neutral and acidic conditions. At 100 and 300 µM
concentrations, BAC had only a slight or no effect on [Ca2+]i at neutral pH. (Fig. 7A and B) Acid
by itself also had only a minimal effect on [Ca2+]i. (Fig. 7D) In contrast, at 500 µM concentration
bile acids induced a reversible increase in [Ca2+]i at pH 7.5, which was more pronounced at pH
5.5. (Fig. 7A and B). Next the source of Ca2+ release was identified. After removing Ca2+ from the
extracellular solution administration of 500 μM BAC, caused a slight increase in [Ca2+]i strongly
suggesting that BAC induces Ca2+ signaling from intracellular sources. (Fig. 7E). Therefore, in
the next step we sought to identify the intracellular organellum from which the Ca2+ releases.
Ryanodine (Ry) and inositol triphosphate (IP3) receptors which mediate the release of Ca2+ from
endoplasmic reticulum were blocked by their specific inhibitors, Ruthenium red (RR) and caffeine
respectively. 10 µM RR had no effect on BAC-induced Ca2+ release in Ca2+ free external solution.
However, 20 mM caffeine completely blocked the effect of BAC on Ca2+ signaling. Administration
of Gd3+ a plasma membrane Ca2+ channel inhibitor decreased the effect of 500 µM BAC on [Ca2+]i
by 58.83 ± 1.3 % (Fig. 7E), indicating that beside the release of Ca2+ from intracellular sources,
bile acids also induce the entry of extracellular Ca2+.
- 27 -
A B
C D
E
0,2
0,5
0,8
1,1
1,4
F3
40
/38
0
0,2
0,5
0,8
1,1
1,4
F3
40
/38
0
Hepes (pH 5.5)
100 µM 300 µM 500 µM
5 min
Hepes (pH 7.5)
100 µM 300 µM 500 µM
5 min
0,0
5,0
10,0
15,0
20,0
25,0
100 µM 300 µM 500 µM
Flu
ore
sc
en
ce (3
40
/380
) %
pH 7.5
N.D. N.D. 0,0
50,0
100,0
150,0
200,0
250,0
300,0
350,0
Control 100 µM 300 µM 500 µM
Flu
ore
sc
en
ce (3
40
/380
) %
pH 5.5
N.D.BAC
BAC
BAC
0,0
2,0
4,0
6,0
8,0
10,0
12,0
14,0
16,0
18,0
20,0
Caffeine Ruthenium Red
Gadolinium
Flu
ore
scen
ce (
340/3
80)
%
500 µM BAC
Ca2+ -free
**
*
N.D.
Figure 7. Effects of bile acids on
[Ca2+]i in CP-A cells. Representative
experimental traces showing the effect
of a 100-, 300-, and 500-μM BAC at
pH 7.5 (A) and pH 5.5 (B) on [Ca2+]i.
Summary data of the bile acid-induced
[Ca2+]i changes at pH 7.5 (C) and pH
5.5 (D). Values are expressed as
percent of basal [Ca2+]i. (E) Effect of
extracellular Ca2+ removal, caffeine
(20 mM), RR (10 μM) and Gd3+ (1
μM) on the rise in [Ca2+]i induced by
500 μM BAC. All experiments were
performed in HEPES-buffered
solution. Data are presented as means
± SE. *P ≤ 0.05 vs. 500 μM BAC; n =
10–21.
- 28 -
8.1.3. Acute effects of bile acids on the activity of ion transporters in EECs
Next, the acute effect of BAC on the activity of ion transporters was examined using the
NH4Cl prepulse technique. Administration of BAC caused a dose-dependent decrease in the
recovery from acidosis in Hepes-buffered solution (Fig. 8A and B), indicating that bile acids
inhibit the activity of NHE in CP-A cells.
A B
C
In order to determine which NHE isoform is involved in the inhibitory effect of bile acids,
the effect of BAC was tested in the presence of the isoform-specific NHE inhibitor, HOE-642. 1
µM HOE-642 decreased the recovery from acidosis from 7.68 ± 1.11 to 1.78 ± 0.2. Administration
6,4
6,6
6,8
7,0
7,2
7,4
7,6
7,8
8,0
8,2
8,4
pH
i
Hepes
NH4Cl NH4Cl NH4Cl NH4Cl
3 min
100 µM BAC 300 µM BAC 500 µM BAC
Recovery from acidosis
in Hepes
0
1
2
3
4
5
6
7
8
9
10
Control 1 µM HOE 1 µM HOE + 500 µM BAC
50 µM HOE 50 µM HOE + 500 µM BAC
J(B
- )
Recovery from acidosis
in Hepes
Figure 8. Effects of bile acids on NHEs in
EECs. (A) representative pHi traces show
the effect of a 100-, 300-, and 500-μM
BAC in HEPES-buffered solution on the
CP-A cell line. Cells were treated with bile
acids 3 min before the pulse started, during
the NH4Cl pulse, and 3 min after the pulse.
(B) Summary of the calculated NHE
activity in CP-A and CP-D cells. (C)
Summary of the effect of 500 μM BAC on
the activities of different NHE isoforms in
CP-A cells in the presence of 1 and 50 μM
HOE-642. Data are presented as means ±
SE. *P ≤ 0.05 vs. control; n = 15–25.
- 29 -
of 500 µM BAC, in the continuous presence of HOE-642, further decreased the acid recovery to
0.56 ± 0.09 (Fig. 8C) Since 500 µM BAC inhibited acid recovery by 77.15 ± 3.2%, and nearly
77% of the total NHE activity is due to NHE1, these results indicate that BAC remarkably inhibits
NHE1, however it also blocks NHE2 activity.
A B
C D
Figure 9. Effects of bile acids on NBC and CBE in EECs. (A) Representative pHi traces show the effect of 100-,
300-, and 500-μM BAC in HCO3−/CO2-buffered solution on the CP-A cell line. Cells were treated with bile acids 3
min before the pulse started, during the NH4Cl pulse and 3 min after the pulse. (B) Summary of the calculated NHE
and NBC activities in CP-A and CP-D cells. The rate of acid recovery [J(B−)] was calculated as described in Fig. 1.
(C) summary data of the calculated rates of pHi recovery from acid load in HCO3−/CO2-buffered solution in CP-A
cells. The effect of bile acids on NBC activity was evaluated in the presence of 50 μM HOE-642. The rate of acid
6,4
6,6
6,8
7,0
7,2
7,4
7,6
7,8
pH
i
HCO3-/CO2
NH4Cl NH4Cl NH4Cl NH4Cl
100 µM BAC 300 µM BAC 500 µM BAC
3 min
Recovery from acidosis
in HCO3-/CO2
Recovery from acidosis
in HCO3-/CO2
0,0
5,0
10,0
15,0
20,0
25,0
Control 50 µM HOE-642 50 µM HOE-642 + 500 µM BAC
J(B
- )
Recovery from alkalosis
in HCO3-/CO2
- 30 -
recovery [J(B−)] was calculated as described in Fig. 1. (D) summary of the calculated CBE activity in CP-A and CP-
D cells. The rate of alkali recovery [−J(B−)] was calculated from the ΔpH/Δt obtained by linear regression analysis of
pHi measurements made over the first 30 s of recovery from the highest pHi level (start point pHi). The buffering
capacity at the start point pHi was used for the calculation of J(B−). Data are presented as means ± SE. *P ≤ 0.05 vs.
control; n = 15–25.
Since Ca2+ plays an essential role in the function of several intracellular processes, we
examined whether the observed inhibitory/stimulatory effect of BAC on ion transporters is
mediated by Ca2+. Pretreatment of the cells with the Ca2+ chelator BAPTA-AM, a significant
decrease was obtained both in the inhibitory and stimulatory effects of 500 µM BAC on the ion
transporters, indicating that the effects of bile acids on ion transporters are Ca2+-dependent (data
not shown).
8.1.4. Chronic exposure of EECs to bile acids
Next, the long-term effects of bile acid treatment on the expression of ion transporters were
assessed. CP-A and CP-D cells were grown to 70-80% confluency and treated with 100 and 500
μM BAC at pH 7.5 and pH 5.5 as described in Material and Methods. 7-days treatment with BAC
significantly increased the expression of NHE1, NHE2, NBC and CBE isoform Slc26a6 compared
to non-treated control cells at pH 7.5 in CP-A cells. (Fig. 10A) The expression of these ion
transporters also increased in CP-D cells, however, significant changes were only detected in the
case of NHE1 and NBC. (Fig. 10B) We have also performed these experiments under acidic (pH
5.5) conditions. In CP-A cells, at acidic pH alone or in combination with bile acids the expression
levels of ion transporters did not change significantly (Fig. 10C) and a decreased cell number was
observed compared to the control groups. In contrast, CP-D cells displayed a significant increase
in NHE1 levels after bile acid treatment at pH 5.5. (Fig. 10D) We have also shown that the
enhanced mRNA levels of NHE1 were associated with significantly increased protein expression.
(Fig. 10E) The Slc26a6 transporter expression also increased in CP-A cells at neutral pH (data not
shown). These data are in accordance with our PCR results. However, in the case of NHE2 and
NBC, there were no significant difference in the protein expression, between the control and the
bile acid-treated group at neutral pH (data not shown).
- 31 -
A B
C D
E
2-Δ
ΔC
T
CP-A CP-D
pH 7.5pH 7.5
2-Δ
ΔC
T
2-Δ
ΔC
T
2-Δ
ΔC
T
pH 5.5pH 5.5
NHE1
Control 100 500
GAPDH
CP-A
pH7.5 pH5.5
NHE1
GAPDH
Control 100 500 µM BAC
CP-D
Control 100 500
pH7.5 pH5.5
Controll 100 500 µM BAC
Figure 10. Expression of ion
transporters in Barrett's cell lines. CP-A
and CP-D cells were treated with different
bile acids for 7 days at pH 7.5 (A and B)
and 5.5 (C and D) and the relative mRNA
expressions of NHE1, NHE2, NBC, and
Slc26a6 were investigated by real-time
PCR. Data are presented as means ± SE.
(E) Western blot analysis for NHE1
protein expression after 100- and 500-μM
bile acid treatments
- 32 -
Finally, we assessed the mRNA expression pattern of ion transporters in human derived
tissue. 14 pairs of normal squamous and BE biopsy samples obtained from patients with known
BE was investigated. (Fig. 11A-C)
A B
C
Using qPCR, increased mRNA expressions of NHE1, NHE2, NBC and Slc26a6 in BE
were found both in intestinal (Fig. 11A) and non-intestinal (Fig. 11B) metaplasia compared to
NHE1 NHE2 NBC Slc26a6
* *
*
* *
*
* *
NHE1 NHE2 NBC Slc26a6
NHE1
MSQ
40X
NC
40X40X
100X100x
40X
100X
NHE2
MSQNC
40X 40X 40X
100X 100X 100X
Figure 11. Expression of ion
transporters in human esophageal
biopsy samples. Box plots show the
relative expression of NHE1, NHE2,
NBC, and Slc26a6 in biopsy
samples derived from intestinal (A)
and non-intestinal (B) metaplasia.
Median values are shown as a
horizontal black bar within each
box. *P ≤ 0.05 vs. normal SE; n = 7.
(C) representative pictures show
IHC staining of NHE1 and NHE2 in
normal esophageal squamous
mucosa and intestinal metaplastic
tissue specimens. Isotype negative
control (NC) was also included to
assess nonspecific staining. SQ,
squamous mucosa; M, metaplasia.
Arrows pointing toward NHE-1 and
NHE-2 staining. Scale bar = 50 μm.
- 33 -
normal epithelium. The protein expression of NHE1 and NHE2 were also investigated by IHC.
Biopsy samples from both intestinal and non-intestinal metaplastic columnar mucosa displayed
strong staining against NHE1 and NHE2 antibodies in contrast to normal SE. (Fig. 11C)
8.2. Role of autophagy in the pathogenesis of GERD and BE
8.2.1. Effects of pharmacologic inhibition of autophagy following acidic stress on ROS
production
First, the role of autophagy in cellular response after acidic stress was evaluated. STR, CP-
A, CP-D and OE19 cell lines were exposed to acidic challenge at pH=3.5 followed by treatment
with 50 M CQ or vehicle control. CQ is a specific inhibitor of autophagy which acts through
blocking the fusion of autophagic vacuoles (AVs) with lysosomes. Six hours post-exposure cells
were stained with DCF or Cyto-ID to determine intracellular ROS levels and autophagic responses,
respectively. DCF and Cyto-ID autophagy fluorescence were quantified by flow cytometry. Flow
cytometry observations were confirmed by confocal microscopy on STR cells (Fig. 12A and C)
Unstained cells in additional plates were maintained until 24 hours post acid exposure, at which
an assay for cell viability was performed.
In both STR and CP-A cells, acid treatment significantly increased ROS levels at 6 hours
post exposure (Figure 12A and B). CQ alone had a mixed impact on cellular ROS levels-increasing
them in STR but not CP-A cells. However, CQ in combination with acid stress induced an
additional significant increase in ROS levels in both STR and CP-A cells compared to cells which
were exposed to acid only (Figure12A and B). Autophagy levels were similarly responsive to these
treatments in STR and CP-A cells. Cyto-ID autophagy levels were increased by all three
conditions: acid exposure alone, CQ, as well as the combined treatments. The increase observed
with CQ is due to the accumulation of blocked AVs (Figure 12C and D).
In both CP-D and OE19 cells, these cellular responses were different. As with the non-
dysplastic cells, the acid treatment led to a significantly increased ROS levels at 6 hours post
exposure in these cell lines (Figure 12B). CQ alone had no significant effect on cellular ROS
levels. However, when CQ was combined with acid stress, there was no additional increase in
ROS levels experienced by either the CP-D or OE19 cells compared to cells which were exposed
to acid alone (Figure 12B).
- 34 -
Figure 12. Effects of autophagy inhibition on intracellular ROS and AV formation after GERD-like acid
exposure. (A) Relative intracellular ROS levels are determined by DCF fluorescence in STR cells at 6 h post
treatment; imaged by confocal microscopy. W: control nonacidic media; A: acid [pH3.5] pulsed; C: CQ treated; AC:
acid pulsed followed by CQ treatment. (B) Summary of DCF fluorescence quantified by flow cytometry in normal
(STR) and BE (non-dysplastic CP-A) cell lines treated as before and measured at 6 h post treatment; n=6 experiments
for each. Significance testing was by one-way ANOVA followed by Tukey's multiple comparison test; a: significantly
differs from control and CQ treated cells; b: significantly differs from acid and CQ treated cells by one-way ANOVA
and Tukey's multiple comparison test; P ≤ 0.05. (C) Relative autophagy induction 6h post treatment with acid or/and
CQ as determined by Cyto-ID fluorescence in STR cells and imaged by confocal microscopy. (D) Summary of Cyto-
ID fluorescence and relative autophagy induction quantified by flow cytometry in representative normal (STR), BE
(non-dysplastic/CP-A and dysplastic/ CP-D) and EAC (OE19) cell lines treated as before and measured at 6 h post-
treatment; n=6 experiments for each. a: significantly differs from control treated cells by one-way ANOVA and
Tukey's multiple comparison test; P ≤ 0.01 0.001. b: significantly differs from acid and CQ treated cells by one-way
ANOVA and Tukey's multiple comparison test; P ≤ 0.05.
Similarly, the quantification of the autophagic response of CP-D cells to acidic stress failed
to show any change in Cyto-ID fluorescent signal after any treatment (Figure 13A), including the
CQ treatment alone. Furthermore, in OE19 cells the exposure to acid significantly reduced this
relative fluorescence signal (Figure 13B). As these observations ran contrary to our expectations,
especially with respect to CQ treatment, we were concerned that there was a problem with the
Cyto-ID fluorescence as a measure of autophagy responses. We therefore examined the Cyto-ID
fluorescent by confocal microscopy in OE19 cells after these same treatments and in parallel
treated LC3-GFP labeled OE19 cells as a second measure of the autophagic response following
the same experimental protocol. LC3 is the most widely used marker for autophagosome and the
examination of GFP-labeled LC3 localization is a very simple and highly sensitive method to
measure autophagic activity in living cells. (6) Unexpectedly, there was a significant difference
between the two methodologies. In untreated OE19 cells, Cyto-ID fluoresces brightly in
- 35 -
cytoplasmic vesicles, while LC3-GFP does not similarly collect (Figure 13C and D). Moreover,
while LC3-GFP vesicles were increased weakly by acid treatment and strongly after CQ treatment,
Cyto-ID fluorescence pattern recapitulated that measured by flow cytometry, with diminished
signal in acid-treated cells (Figure 13C and D).
Figure 13. Cyto-ID fluorescence does not correlate with LC3-GFP vesicle levels in OE19 cells. (A) and (B)
Summary of Cyto-ID fluorescence quantified by flow cytometry in representative dysplastic BE (CP-D) and EAC
(OE19) cell lines treated as before and measured at 6 hours post-treatment. N=control nonacidic media; A=acid [pH
3.5] pulsed; C=CQ treated; AC=acid pulsed followed by CQ treatment. Significance testing was by one-way ANOVA
followed by Tukey’s multiple comparison test; n=6 experiments for each. ** not significantly different by one-way
ANOVA p=0.2635. a= significantly differs from control and CQ treated cells; adjusted P ≤ 0011. ***= not
significantly differs from control treated cells; adjusted p=0.99. (C) Relative autophagy induction 6 hours post
treatment with acid or/and CQ as determined by Cyto-ID fluorescence in STR cells and imaged by confocal
microscopy. N=control nonacidic media; A=acid [pH 3.5] pulsed; C=CQ treated; AC=acid pulsed followed by CQ
treatment. (D) Cells imaged by epifluorescence microscopy to visualize fluorescent AV puncta in LC3-GFP
expressing OE19 cells after acid and/or CQ treatment.
- 36 -
8.2.2. Acidic challenge diminishes cell viability after pharmacologic inhibition of autophagy
by CQ
In a final study, we were interested whether the autophagic flux following acid treatment
gives any functional benefit to the cells demonstrated by the survival advantage provided by an
autophagic response. 7AAD staining was used as a highly quantitative flow cytometry approach
to assess cell death. 7AAD+ dead cells were quantified 24 hours after treatment with acid, CQ, or
the combination of acid and CQ, in all four cell. Cell death after acid treatment was increased in
three of four cell lines (STR, CP-A, and CP-D), but this was most significant for CP-D cells (Figure
14A, B, and C). STR cells were uniquely sensitive to CQ, with a nearly 5-fold increase in 7AAD+
dead cells after 24 hours of treatment. Most important, for all cell lines examined, the combination
of acidic stress and CQ treatment led to a very significant increase in 7AAD+ dead cells at 24
hours (Figure 14A, B, C, and D). In STR, CP-A, and OE19 cells, the combination of acidic stress
and inhibition of autophagy synergized and led to greater level of cell death than the sum of the
individual treatments (Figure 14A, B, and D).
Figure 14. Inhibition of autophagy increases cell death after GERD-like acid exposure. Flow cytometric
quantitation of 7-amino-actinomycinD (7AAD) cell staining at 24 h in (A) STR cells, (B) CP-A cells, (C) CP-D cells,
and (D) OE19 cells. 7AAD+ cells are expressed as fold-increased over non- acid treated control cells. N: control
nonacidic media; A: acid pulsed [pH3.5]; C: CQ treated; AC: acid pulsed followed by CQ treatment; n 1⁄4 6
experiments for each. Significance testing was by one-way ANOVA followed by Tukey's multiple comparison test;
P-values adjusted for multiple comparisons are reported. a: significantly differs from acid and CQ treatments, P=
0.0018. b: significantly differs from CQ treatments, P 0.015. c: significantly differs from acid and CQ treatments, P=
0.028.
- 37 -
8.3. Modeling esophagitis using 3D organotypic culture system
8.3.1. The organotypic culture environment sustains immune cells and permits their
normal activation when stimulated by cytokines
OTC systems allow for the co-culture of immortalized normal human esophageal epithelial
cells together with primary human fibroblasts in 3D tissue reconstructions that recapitulates
normal esophageal epithelial morphology and differentiation (Figure 15 A and B). (64) In order to
develop a human in vitro model of esophageal inflammatory conditions human PBMCs were
included in the collagen/Matrigel extracellular matrix at the initiation of the culture, prior to the
establishment of the epithelial cell layer (Figure 15A and C). To insure immune cell viability
during the 14 day culture period, IL-2 was added to the cell culture media. PBMCs remained
viable throughout the culture period, as indicated by CD45+ cells in the collagen matrix/stromal
compartment of the OTC cultures (Figure 15C). Interestingly, no spontaneous activation of
immune cells was observed, demonstrated by the absence of lymphocyte proliferation (Figure
15D) and cytokine production (data not shown), despite the fact that the PBMCs, fibroblasts and
epithelial cells were derived from different donor source.
In order to induce a robust TH1 acute inflammatory response, we elected to include the
potent pro-inflammatory cytokines IL-7 and IL-15 in the culture media. IL-7 and IL-15 are both
tissue-derived cytokines most abundantly expressed by stromal and epithelial cells, including
keratinocytes. (80-83) Both cytokines are potent inducers of TH1 acute inflammatory cytokines
including interferon- in T-cells and monocytes, and both have profound effects on T-cell survival
and proliferation, including helper (CD4+) and effector (CD8+) T-cells and NK-cells, during all
phases of T-cell development. (83, 84) Unlike IL-2 alone, these three cytokines together supported
the induction of an acute inflammatory response in the OTC-PBMC co-culture environment.
- 38 -
Figure 15. Modeling esophageal inflammation. (A) Illustration of the standard OTC method (left), as well as the
modification introduced by incorporating PBMCs into the collagen matrix to model inflammation (right). (B)
Representative pictures show H&E staining of normal human esophageal keratinocytes grown under standard OTC
conditions. (C) CD45 IHC staining of an OTC culture with human PBMCs and IL-2 added. CD45+ staining indicates
the presence of immune cells in the collagen matrix. Inset: higher power magnification of CD45+ cells. (D) IHC
staining for the cell proliferation marker Ki-67 in an OTC+PBMC culture. Ki-67+ cells noted in the epithelium (Brown
arrows) but not in stromal cells. Inset: higher power magnification of Ki-67 negative stromal cells. (E) IHC staining
for the cell proliferation marker Ki-67 in an OTC+PBMC+IL culture. Ki-67+ cells noted in both the epithelium and
stromal cells (Brown arrows). Inset: higher power magnification of Ki-67+ stromal cells. Scale bar represent 50 μm
The addition of IL-7 and IL-15 induced a very significant increase in the number of CD45+
immune cells in the OTC-PBMC cultures (Figure 16A, right panel). This effect was not observed
when PBMCs were co-cultured with only IL-2 (Figure 16A, center panel). While some of the
increased numbers of CD45+ cells might be due to increased cell survival with IL-7 and IL-15, a
portion of the increase was due to immune cell proliferation, as indicated by the Ki67+ stromal
cells in the OTC+PBMC+IL cultures (Figure 15E) but not the OTC+PBMC control (Figure 15D).
In addition to the increased numbers of PBMCs, there was a noticeable infiltration of lymphocytes
into the OTC epithelium, characteristic of esophagitis, with the addition of the pro-inflammatory
interleukins (Figure 16B). A general characteristic of immune cell activation is the increased
production of cytokines. We were unable to directly detect the canonical TH1 cytokine IFN-in
cell culture media from the OTC+PBMC+IL cultures despite multiple attempts (data not shown).
We suspect this is due to the dense OTC collagen matrix in which the PBMCs are maintained in,
- 39 -
as we readily detect increased IFN-levels in the cell culture media when PBMCs are cultured in
a soft Matrigel matrix and exposed to the same TH1 pro-inflammatory cocktail IL-2, -7, and -15
(data not shown). As a surrogate marker, we examined the overlying epithelium in the OTC
cultures for expression of interferon regulatory factor 1 (IRF-1), a member of the interferon
regulatory transcription factor family and a well-known target gene of IFN-signaling. (85) We
observed a highly significant increase in the levels of IRF-1 mRNA and protein detected in the
epithelium from the OTC+PBMC+IL cultures (Figure 16C and D). However, IRF-1 mRNA and
protein were not increased in the epithelium from control cultures treated either with the TH1
promoting interleukins or with PBMCs alone (Figure 16C), indicating this response was specific
to the OTC+PBMC+IL cultures. Together these findings establish that human PBMCs
incorporated into the OTC culture environment remain viable and can proliferate and produce IFN-
when provoked by pro-inflammatory cytokines.
Figure 16. Organotypic culture system allows the proliferation and activation of immune cells. (A)
Representative pictures show IHC staining for CD45+ cells, a marker of leukocytes, in OTC control (left panel),
OTC+PBMCs (center panel), and OTC+PBMC+IL (right panel) cultures. Scale bar represent 50 μm. Inset: Arrow
pointing towards positive CD45+ cells in the collagen matrix of the OTC+PBMC+IL culture. (B) Representative H&E
staining shows the presence of infiltrating lymphocytes in the OTC+PBMC+IL epithelium (black arrows). Inset:
Higher power view of infiltrating lymphocytes. (C) qPCR quantitation of relative mRNA expression of IRF-1 in the
epithelium of the OTC cultures. Data are presented as mean±SEM. n=3 for OTC+PBMC and OTC+IL; n=7 for OTC
control and OTC+PBMC+IL. * significantly differs from all others by ANOVA and TUKEY Rank Mean testing,
P≤0.05 (D) Representative Western Blot analysis for IRF-1 protein levels in the epithelium from the OTC control and
OTC+PBMC+ILs-treated cultures. Blots were stripped and reprobed for the loading control tubulin.
- 40 -
8.3.2. Inflammatory OTC environment induces changes in epithelial morphology
We next sought to identify the effects of activated immune cells on the overlying EECs.
To control for the effects of IL-2, IL-7, and IL-15, or the effects of PBMCs alone, OTC cultures
with interleukins or immune cells alone were established, in addition to the combined
OTC+PBMCs+ILs. These OTC cultures were fixed and embedded and then stained with
hematoxylin and eosin to examine for morphologic changes in the epithelium. Cultures with
cytokine-cocktail treated PBMCs displayed a significant increase in epithelial thickening, both in
the basal and suprabasal compartments (Figure 17A-D). This doubling of epithelial thickness in
the OTC cultures was highly significant and confirmed by measurements across multiple cultures
(Figure 17E).
Figure 17. Morphological effects of activated immune cells on esophageal epithelium. Representative pictures of
hematoxylin-eosin staining show the morphological differences of OTCs under different culture conditions, including
(A) Control OTC, (B) OTC with only PBMCs added (OTC+PBMC), (C) OTC with only IL-2, IL-7, and IL-15 added
(OTC+IL), and (D) OTC with both PBMCs and the interleukin cocktail (OTC+PMBC+IL). Inset: significant
epithelial thickening was observed together with hyperkeratinization (blue arrow) and regenerative keratin pearls
(black arrow) only in the OTC+PBMC+IL culture. These changes were not present in control cultures. Scale bar
represent 50 μm. (E) Quantification of epithelial thickness in microns, measured in 4-5 regions on each section and
from three different tissue sections per culture, three separate cultures in total for each treatment. Data are presented
as mean±SEM. n=3 for all cultures. * and ** significantly differs from all others by ANOVA and TUKEY Rank
Mean testing, P≤0.05
- 41 -
8.3.3. The pro-inflammatory environment modeled with the OTC culture system alters
cell proliferation and cell death in the epithelium
The thickening of the OTC epithelium in the acute inflammatory environment suggested
there might be changes in epithelial cell proliferation. We examined for changes in proliferative
rates by staining for the proliferation marker Ki-67 in the epithelial cell layer. Basal epithelial cell
proliferation in the OTC epithelium was noticeably enhanced in the OTC cultures with PBMCs
and the interleukin cocktail when compared to controls (Figure 18A). There was a significant
increase in Ki-67+ cells, as compared to controls, when quantified across multiple cultures (Figure
18B).
Figure 18. The acute inflammatory environment induces OTC epithelial cell proliferation. (A) Representative
Ki-67 immunohistochemical staining in control OTC culture and the OTC culture treated with both PBMCs and the
proinflammatory IL-cocktail. Scale bar represents 50 μm. (B) Quantitation of Ki-67 positive staining. The number of
Ki67+ cells was expressed as a percentage of the total number of keratinocytes in a counted field. Data are presented
as mean±SEM. n=3 cultures per condition, with 500 cells counted per culture. * Significantly differs from OTC
control by unpaired Student’s T Test, P≤0.0001.
This elevated proliferative rate was off-set in part by increased epithelial cell apoptosis.
This increased level of cell death was confirmed and quantified by TUNEL staining. While there
were rare TUNEL+ cells in the control OTC cultures (Figure 19B), there was significantly
increased numbers of TUNEL+ epithelial cells in the epithelium of the OTC cultures with the
acute-inflammatory environment (Figure 19B). Counts of apoptotic cells over multiple
OTC+PBMC+IL cultures revealed a 10-fold increase in TUNEL+ cells as compared to control
OTC cultures (Figure 19C). Together these findings support the conclusion that an acute
inflammatory environment in the OTC culture system induces epithelial responses including
increased epithelial cell apoptosis and proliferation.
- 42 -
Figure 19. Epithelial cell apoptosis is significantly increased in OTC cultures treated with PBMCs and IL-2,
IL-7, and IL-15. (A) Representative epiflourescence images of combined nuclear DAPI (blue) and the apoptosis
marker TUNEL (red) staining in OTC control and OTC+PBMC+IL cultures. (B) Quantitative analysis of TUNEL
positive cells. TUNEL positive cells were expressed as a percentage of the total number DAPI stained cells in a field.
Data are presented as mean±SEM. n=3 cultures per condition, with 500 cells counted per culture. * Significantly
differs from OTC control by unpaired Student’s T Test, p≤0.005.
BA
- 43 -
9. DISCUSSION
In the first part of this thesis we characterized the main pHi regulatory mechanisms of the
EECs. The preservation of physiological pHi is critical for cell survival and it is mediated through
intracellular buffers and ion transporters of the membrane. Ion transporters are part of the
esophageal epithelial defense and play crucial role in the protection of gastric and bile acid induced
esophageal injury. In contrast to the SE, publications in connection with the ion transport
mechanisms of the metaplastic mucosa are lacking, therefore the function and expression of these
transporters were investigated on Barrett’s derived cells and tissues.
Using functional and molecular biology methods, we determined that Barrett’s derived
EECs display three main functionally active ion transporters: the NHE, the NBC and the CBE
isoform Slc26a6, similarly to other epithelial cells in the gastrointestinal tract. (75, 86, 87) Using
NHE isoform specific inhibitor HOE-642, we determined that NHE-1 and NHE-2 are participating
in the restoration of pHi which is rather attributable to NHE-1. This finding was in accordance with
Goldman et al. who found that 60% of pHi recovery is mediated through NHE-1. (44) Furthermore,
we determined that NHE and NBC contribute equally to the alkalization of CP-A and CP-D cells.
To determine the effects of bile acids, we designed a bile acid mixture mimicking the bile
acid composition of the refluxate under pathophysiological conditions. Since bile acids are known
to induce many intracellular processes like Ca2+ signaling, we investigated the effect of BAC on
[Ca2+]i both under neutral (pH=7.5) and acidic (pH=5.5) conditions. We found that administration
of bile acids induced dose-dependent Ca2+-elevation in the cells which was more pronounced under
acidic condition. Our findings were in agreement with observations of other laboratories that
demonstrated that exposure CP-A cells or mouse EECs to DC or acidic media induced intracellular
Ca2+ elevation. (88, 89) Furthermore, we investigated the potential source of intracellular Ca2+-
release. We showed that caffeine, an inhibitor of IP3-mediated Ca2+ responses, completely
inhibited the bile acid-induced Ca2+ signaling in the absence of extracellular Ca2+, suggesting the
involvement of IP3 receptors in the bile acid-induced Ca2+ release, similarly to colonic crypts,
hepatocytes, or pancreatic ducts and acini. (71, 86, 90, 91) Gd3+, a known inhibitor of plasma
membrane Ca2+ entry channels, strongly blocked the bile acid-induced Ca2+ signaling indicating
that bile acids also promote the influx of extracellular Ca2+.The exact mechanism by which bile
acids induce the entry of extracellular Ca2+ is not known. In rat hepatocytes, bile acids directly
- 44 -
stimulate store-operated Ca2+ channels on the plasma membrane (92); however, further
investigations are necessary to identify those Ca2+ channels that contribute to the effect of bile
acids on the esophagus.
Since preservation of pHi is dependent upon the activity of ion transporters in the next
series of experiments we investigated the acute response of the previously characterized ion
transporters upon exposure to BAC. Administration of BAC dose dependently decreased the
activity of NHEs (both NHE1 and NHE2), whereas stimulated the activities of NBC and Slc26a6
in CP-A cells. Inhibition of NHEs probably contributes to the acidification of the CP-A cells. In
contrast, the acidification and the consequent cell damage is prevented by the increased activity of
the HCO3− import system through the NBC. In addition, the efflux of HCO3
− through the
Cl−/HCO3− exchanger, Slc26a6 also plays an important role in the protection of the cells by the
neutralization of the cell environment in the surface mucus layer. We speculate that the increased
activities of NBC and Slc26a6 probably compensate the decreased NHE activity and therefore try
to maintain the acid/base equilibrium of the cells. Interestingly, administration of BAC stimulated
NHE activity in CP-D cells. This difference to CP-A cells can be explained by the advanced stage
of CP-D cells. It has been described earlier that dysplastic Barrett's mucosa has more severe and
prolonged acidic and biliary reflux exposure. (93) Furthermore, it has also been observed that CP-
D cells are more resistant to GERD-like stimuli compared with CP-A cells. (94) The mechanism
for this alteration and its potential physiological role cannot be explained by the present studies
and are areas of focus for future work.
The underlying mechanism by which bile acids exert their stimulatory/inhibitory effects
has also been investigated. Previous studies have demonstrated that the effect of bile acids on ion
transporters is mediated by transient elevation of [Ca2+]i .(71, 86) Our results have shown that
chelation of [Ca2+]i by BAPTA-AM almost completely abolished both the inhibitory and
stimulatory effect of BAC on ion transporters. Although we have not studied the mechanism by
which Ca2+ mediate the effect of bile acids on CP-A cells, we propose that the activation of Slc26a6
is connected to the activation of Ca2+-activated ion channels, such as Ca2+-activated Cl− channels
or K+ channels, as demonstrated in other epithelia. (95-97) In contrast to Slc26a6, high levels of
[Ca2+]i strongly inhibited NHE activity. Previous studies on rabbit ileal brush-border membrane
and renal NHE containing proteoliposomes have demonstrated that the phosphorylation of specific
proteins by the Ca2+/calmodulin cascade results in a robust blockade of NHE. (98, 99) Taken
- 45 -
together these data indicate that the increased levels of Ca2+ probably do not directly modulate the
activity of ion transporters; however, further investigations are needed to identify those
intracellular signaling pathways or molecules that are involved in this process.
In the next step, in order to mimic chronic bile acid challenge, we performed a 7-day
treatment with BAC under neutral and acidic conditions to investigate the role of ion transporters
in cellular adaptation. Following treatment the mRNA expression of all of the investigated
transporters in CP-A cells and the mRNA expression of NHE1 and NBC in CP-D cells were
increased under neutral pH. In contrast, the expression of the transporters did not change
significantly under acidic conditions in CP-A cells; moreover, the cell number dramatically
decreased. In contrast, the expression of NHE1 significantly increased in CP-D cells at pH 5.5. We
could also confirm the increased expression of NHE1 at protein level. To further extend these
studies we investigated the expression profile of these transporters in human derived biopsy
samples from the normal squamous and metaplastic mucosa of BE patients. In Europe, BE is
characterized by the presence of macroscopically visible metaplastic columnar epithelium. (100)
In contrast, in the United States only the intestinal type of metaplasia is considered to be BE. (13)
Thus we divided our samples into intestinal and non-intestinal groups and analyzed them
separately. NHEs, NBC, and Slc26a6 displayed higher mRNA levels in both intestinal and non-
intestinal metaplasia compared with normal tissue. Increased protein expression of NHE1 and
NHE2 was also confirmed in BE. These results are consistent with the report by Goldman et al.
(44) that demonstrated upregulation of NHE-1 in BE compared with normal epithelium both at
mRNA and protein levels in biopsy samples and cell lines. Moreover, we observed strong apical
staining for NHE-2 in BE biopsy samples, similarly to findings of other laboratories in various
tissues. (101-103) These findings support our hypothesis that the overexpression of ion
transporters is probably a defensive or adaptive mechanism by which the cells try to compensate
the toxic effects of bile acids.
In the second part of the thesis we investigated the potential contribution of autophagy in
the pathogenesis of BE. Autophagy is a highly conserved cellular mechanism which degrades the
unnecessary cellular components by lysosome dependent fashion. Pro-survival function of
autophagy has an adaptive role and activated upon cellular stress like starvation or oxidative stress
in normal, healthy tissue. (48, 50, 51) However, in tumor tissue autophagy can contribute to
increased cell survival: tumor cells located within a hypoxic and nutritionally starved centers
- 46 -
typically utilize autophagy to enhance tumor cell survival. (48) This feature has an important
therapeutic implication as well: pharmacological autophagy inhibitors including CQ and its
derivatives have been effective in enhancing the cytotoxic effects of radiation or chemotherapy
and improving survival in several trials. (104, 105) However, the role of autophagy in premalignant
diseases like BE is less characterized. Several previous observations have demonstrated that
elevated level of ROS in response to GERD is a key step driving BE onset and progression to
EAC. (27, 55-57) Given this well-established relationship between elevated ROS and an increase
in autophagy steady- state levels, suprisingly this pathway has not yet been extensively studied in
GERD and BE. To date, there is a single published paper on this subject, and the focus of this work
is on Beclin-1 and how its loss correlates with progression to EAC. (58) In present study we aimed
to determine the functional role of autophagy in various esophageal cell lines. We found that acid
injury alone, in the absence of bile acids, was sufficient to induce autophagic response in all tested
cell lines. Furthermore, we determined that autophagy inhibition with CQ further increased
intracellular ROS level after acid pulse treatment in non-transformed cell lines (STR and CP-A).
This significant increase is consistent with autophagy acting to reduce intracellular ROS stress
after an injury. (106) However, this significant increase was not observed in dysplastic cells (CP-
D and OE-19), suggesting that these cells do not utilize autophagy to manage oxidative stress
induced by an acidic environment. Most significantly, we observed that the inhibition of autophagy
after an acidic insult leads to significantly greater cell death at 24h in all cell lines tested. This
suggests two important role for autophagy in esophageal cells in response to an acid insult: the
modulation of the oxidative stress and enhancing cell survival. One unexpected finding from our
study is that in dysplastic CP-D and OE19 cells, Cyto-ID, and LC3-GFP appear to mark different
vesicle subsets. This is the only explanation available to explain the discordance between these
two well-established approaches to quantifying autophagy vesicle content of cells. We tend to
favor the LC3-GFP results as being more representative of the autophagy response, given that
control OE19 cells by observed by transmission electron microscopy had vesicles but these were
not double-walled and did not contain cellular debris (data not shown). Thus the Cyto-ID dye
identifies these vesicles based on a shared biochemistry with autophagosomes. However, quite
unexpectedly only in OE19 cells, the Cyto-ID signal was lost after acid treatment. Together these
findings do suggest autophagosome biochemistry may be different in the dysplastic cell line. The
- 47 -
mechanism for this alteration, and the physiologic role it may play, cannot be determined by the
present studies and needs to be further explored.
Research into GERD has previously been limited by the availabilty of suitable laboratory
approaches to model this conditions. Much of the past work has relied upon the availability of
human patient biopsies (107), which are a precious commodity and largely suitable for descriptive
studies only. Commercially available immortalized cell lines, including representing normal
squamous, Barrett’s, and EAC, are maintained under 2D culture conditions and are the most
convenient and widely used in vitro tools. (60-62) However, 2D cultured immortalized cell lines
can not reflect appropriately the complex interactions between epithelial cells and their
microenvironment. (108) Animal models are another important tools of biomedical research. An
ideal animal model should represent the human etiology, pathogenesis and molecular features of
the disease and these models leave significant amounts of unmet needs. Surgical models such as
esophagojejunostomy, esophagoduodenal anastomosis, and esophagogastroduodenal anastomosis
in rats (109-114) and less frequently in mice (115) have been the primary methods used to mimic
reflux induced mucosal injury. However, the use of rats has been limited due to the paucity of
genetically modified rat transgenic lines which precludes the conduction of mechanistic genetic
studies. In contrast, while there are an abundance of suitable transgenic mouse lines for
mechanistic studies (13, 116-118) the surgical procedure in mice is technically much more
challanging, limiting its broader application. A transgenic mouse model for esophagitis that
progresses to BE and EAC has been described. (13) It utilizes an Epstein-Barr virus L2 promoter
to over-express human IL-1β in the oral cavity, esophagus, and squamous forestomach of mice.
These L2-IL-1β mice develop a chronic (119) inflammatory esophagitis by 3 months that is
followed subsequently by the development of a columnar metaplasia with intestinal features and
later dysplasia and cancer. However, in this transgenic line the IL-1β is constitutively expressed.
This is therefore a model of chronic inflammation rather than suitable for studies of the effects of
an acute inflammatory environment on epithelial cell responses. Thus, the limitations of the
aforementioned in vitro and in vivo approaches pose a significant constraint to the field. Disease
models for GERD and esophagitis based on 3D human cell and tissue culture systems that
recapitulate in vivo growth and differentiation in inflammatory-associated environment would
enhance our understanding of disease progression and improve our ability to test for disease
prevention strategies.
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Therefore, in the third part of the thesis we further expanded the original protocol of OTC
in order to model esophageal inflammation and get a better insight into the interactions between
the esophageal epithelium and the acute inflammatory environment. We found that OTC is an
appropriate culture system to maintain immune cell viability and cytokine responsiveness, and we
demonstrated that human PBMCs incorporated into the OTC culture respond approapriately to
pro-inflammatory cytokines. Moreover, the epithelial responses to our inflammatory environment
are in accordance with previous descriptions of in vivo responses to reflux esophagitis. (120) We
observed changes in epithelial morphology in our OTC cultures, including increased epithelial
thickening, enhanced basal cell proliferation, elevated levels of epithelial cell apoptosis, and
hyperkeratinization. Biopsy samples from reflux esophagitis patients have similarly displayed
basal cell hyperplasia, and increased epithelial cell apoptosis in the setting of an immune cell
infiltration. (120) Similar findings has been described in rat reflux models as well. (121) Our
observations here imply that a significant portion of these epithelial responses in GERD
esophagitis are immune cell mediated, rather than directly caused by the actions of the gastric
relfuxate, and therefore support this recently proposed alternative mechanism for the tissue injury.
(32)
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10. CONCLUSIONS AND NEW RESULTS
In the first part of the thesis we characterized the role of ion transport mechanisms in
metaplastic EECs. Our findings further confirm the notion that metaplastic change of the
normal SE to BE may be an adaptive process to regulate pHi after exposure to gastric and
bile acid. Our study may help to better understand the metaplastic esophageal response to
injury and the role of ion transporters in this process. We believe that pharmacological
activation of ion transporters increases epithelial resistance in an acidic environment and
therefore may protect the esophageal mucosa against the injurious bile acids.
In the second part of the thesis we demonstrated that autophagy is activated in response to
GERD-like acidic stress where it functions to reduce intracellular oxidative stress and
improve cell survival. We hypothesize that autophagy may be a novel therapeutic target in
BE that deserves to be explored. Our work exploring the activity of autophagy in BE and
EAC is thus important both for mechanistic insights as well as the potential application of
novel therapeutic agents to intervene in BE and EAC onset and progression.
In the final part of the thesis we demonstrated that OTC is a useful tool to model esophageal
inflammation since it can reproduce the characteristics of esophagitis in a well-controlled
in vitro setting. In conclusion, our results further highlight the importance of immune-cell
mediated esophageal injury and supports the further use of this new platform to
charachterize the underlying molecular events in inflammation-induced esophagitis, BE,
and carcinogenesis .
In summary, our studies provided important data on the pathogensis of GERD and its
complications, which may both enhance our understanding of these conditions and open new
perspectives for further research.
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11. ACKNOWLEDGEMENTS
I would like to thank all of the people who have helped and inspired me during my work.
I am grateful to Prof. Dr. Andras Varro, the head of Department of Pharmacology and
Pharmacotherapy and to Prof. Dr. György Abrahám and Prof. Dr. Tibor Wittmann, the current
and former head of the First Department of Medicine, who gave me the opportunity to work at
their Departments.
I would like to say thank you to my supervisors, Dr. Viktoria Venglovecz and Dr. Andras
Rosztoczy for their help during my Ph.D. studies.
I also would like to say thank you to Prof. Dr. Peter Hegyi and Prof. Dr. Zoltan Rakonczay Jr.
besides the opportunity to do my research in their laboratory, for their trust, help, support and
constructive advices. In addition, I would like to thank my colleagues: Dr. Mate Katona, Dr.
Krisztina Toth, Dr. Anita Balazs, Zsolt Balla, Dr. Balázs Kui, Dr. Eszter Kormanyos,
Tamara Madacsy, Dr. Jozsef Maleth, Dr. Petra Pallagi, Dr. Eszter Vegh, for all the help and
entertainment they provided. This work would not have been possible to accomplish without the
assistance and work of Tünde Pritz, Miklosne Arva, Rea Fritz, Edit Magyarne Palfi and the
assistans of the Endoscopy laboratory. My special thanks goes to Zoltanne Fuksz whose
continuous attention and care established the proper conditions of our tissue culture laboratory.
I would like to express my gratitude to Dr. John P. Lynch from the University of Pennsylvania,
Perelman School of Medicine, who was a great mentor of me during my staying in his lab.
Last but not least, my deepest gratitude goes to my parents and Norbert Pardi for their love,
support and encouragement; this dissertation would have been impossible to accomplish without
their help. I would like to dedicate this thesis to them.
This work was supported by the National Development Agency grants (TAMOP-4.2.2.A-
11/1/KONV-2012-0035, TAMOP-4.2.2.A-11/1/KONV-2012-0073, TAMOP-4.2.2- 11/1/KONV-
2012-0052), and as part of the NCI BETRNet program (CA163004-AR, GG, GF, and JL), and a
NIH career development award to JPL (OD 012097). This work was also supported by core
facilities (Molecular Pathology and Imaging, Cell Culture, and Molecular Biology Cores) of the
Center for Molecular Studies in Digestive and Liver Disease at the University of Pennsylvania
(P30-DK050306 and PO1 CA098101) and a scholarship from the Rosztoczy Foundation.
- 51 -
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